SHORT NOTES FOR B. SC. III SEMESTER – VI (BOTANY)

Transformation Experiment by Griffith (05 Marks)

1. In 1928 by Frederick Griffith, conducted experiments suggesting that bacteria are capable of transferring genetic information through a process known as transformation

2. Griffith used two strains of Pneumococcus (which infects mice), a type III-S (smooth) and type II-R (rough) strain.

3. Infection by S – Strain causes Pneumonia to mice and kills it, whereas, R- strain could not cause pneumonia.

4. Heat killed S – strain not causing disease But mixing of R – strain and Heat killed S – strain causes disease

5. Griffith concluded that the type II-R had been "transformed" into the lethal III-S strain by a "transforming principle" that was somehow part of the dead III-S strain bacteria.

Hershey-Chase experiments (05 Marks)

1. The Hershey-Chase experiments were a series of experiments conducted in 1952 on the T2 phage, a virus.

2. The phage consists only of a protein shell containing its genetic material.

3. The phage infects a bacterium by attaching to its outer membrane and injecting its genetic material

4. In a first experiment, they labeled the DNA of phages with radioactive Phosphorus-32 and proteins labeled the phages with radioactive Sulfur -35.

5. The radioactivity found in newly formed phages was in DNA P 32 and not in proteins

6. This experiment has proved that the genetic material was DNA and not protein Hershey shared the 1969 Nobel Prize in Physiology or Medicine for this “discoveries concerning the genetic structure of viruses.”

Chemistry of DNA (12 or 05 Marks)

Fisher – 1880 suggested only Purine as component of DNA. But Levene – 1910, discovered that DNA is Composed of Four Different Ribonucleotides (A,T,G,C).

The molecular structure of DNA can be imagined as a zipper with each tooth represented with opposite teeth forming one of two pairs, either A=T or G≡C.

1. DNA is made up of subunits which scientists called nucleotides.

2. Each nucleotide is made up of : A. Ribose sugar, B. phosphate and C. Nitrogenous Bases.

3. There are 4 different bases in a DNA molecule:

adenine and guanine (purines) cytosine and thymine (a

pyrimidine)

4. The number of purine bases equals the number of

pyrimidine bases

5. The number of adenine bases equals the number of

thymine bases

6. The number of guanine bases equals the number of

cytosine bases

7. The basic structure of the DNA molecule is helical, with the bases being stacked on top of each other

Watson Crick Model of Double Helical Structure of DNA (12 or 05 Marks)

By 1953 they had built a model which incorporated all known features of DNA, and proposed the double helix

structure which is commonly referred to as the Watson-Crick model of DNA.

Crick, Watson and Wilkins were awarded a Nobel prize for this work in 1962.

Based upon the above facts, Watson and Crick proposed the famous DNA structure model. The important

features of this model are:

1. The DNA molecule is a double helix with single polynucleotides (phosphates, sugar, base) running in

opposite directions

2. The double helix is right-handed.

3. The double helix has two different grooves. The helix is not absolutely regular. A major (-22A) and a

minor (-12A) groove can be distinguished.

4. The nitrogenous bases are stacked towards the

inside of the helix.

5. Bases of the two polynucleotides interact by

hydrogen bonding. An adenine residue in one of

the polynucleotides is always adjacent to a

thymine in the other strand; similarly guanine is

always adjacent to cytosine.

6. Ten base pairs occur per turn of the helix.

7. The double helix executes a turn every ten base

pairs (abbreviated as 10 bp).

8. The height or pitch of the helix is 34 A0. The

bases are stacked one on top of the other like a

pile of plates.

9. The space between the two base pairs is 3.4 A0 and has an angle of 36°.

10. The diameter of the helix is 20 A0.

DNA replication in Eukaryotes (12 or 05 Marks)Replication is the production of copies of the DNA (Genetic Material) during cell division. Modes: 1. Conservative model: Both parental strands stay together

after DNA replication.2. Semiconservative model: When the double stranded DNA

helix was replicated, each of the two double stranded DNA helices consisted of one strand coming from the original helix and one newly synthesized.

3. Dispersive model: Parental and daughter DNA are interspersed in both strands following replication.

The widely accepted mode of replication is Semiconservative mode of replication.

Mechanism of Replication:

Replication of both the strands of DNA will be carried out in different way:

Continuous Replication (Leading 3’to 5’) Discontinuous Replication (Lagging 5’ to 3’) with Okazaki Fragments

Steps in replication –I. Unwinding DNA: The unwinding of DNA is carried out by the enzyme Helicase, which breaks the

hydrogen bonds between the strands and separate them.II. Primer Formation: The initiation of new strand sysnthesis is carried out by the enzyme called as RNA

primaseIII. Elongation: Once the RNA primer initiated the replication, the DNA polymerase takes over the charge of

polymerization and it polymerize DNA for both the strands.• Parental strand with 3’ to 5’ and new

strand with 5’ to 3’ has continuous formation of the new strand called as leading strand.

• The parental strand with direction of 5’ to 3’ cannot continuously polymerized. Therefore, this strand will be copied in fragments called as Okazaki Fragments.

IV. Proofreading: If any mismatched base is inserted in the new strand by mistake, the DNA polymerase I worked as exonuclease enzyme to remove the incorrect base.

V. Primer Removal: DNA polymerase III removes the RNA strand and synthesize the DNA strand in place of removed RNA.

VI. Ligation: The lagging strand used to be synthesized in patches and it creates gaps in the Okazaki Fragments. These gaps are sealed by enzyme DNA ligase.

NUCLEOSOME (05 Marks)

Each DNA strand wraps around groups of 8 (4 X 2) small

protein molecules called histones, forming a series of bead-

like structures, called nucleosomes.

The histones are a family of small, basic proteins present in

all eukaryotic nuclei. The five major types of histone

proteins— termed H2A, H2B, H3, and H4 with H1 Linker

Dimensions: Height – 6 nm, and Diameter – 11 nm

Nucleosomes contain 147 base pairs of DNA wrapped slightly less than two turns around the protein

core.

The length of the linker DNA is more variable among species, ranging from about 15 to 55 base pairs.

Histones are forming heterodimer: H 2 A and H 2 B forms two Dimers and H 3 and H 4 forms one

Tetramer

SOLENOID (05 marks)

In case of DNA Packaging the solenoid defines “the packing of DNA as a 30 nm

fiber of chromatin and results from the helical winding of at least six nucleosome

strands”.

These six nucleosomes brought closer by linker H1 histone.

SATELITE DNA

Satellite DNA consists of highly repetitive DNA, and is so called because

repetitions of a short DNA sequence.

When genomic DNA is separated on a CsCl2 Density Gradient Centrifuge,

such repetitive DNA appears as a second or 'satellite' band, hence known as

Satellite DNA.

REPETITIVE DNA

The same sequence of DNA (e.g. AAATTTAAATTT) is present at 1000s time in the same genome is called as

REPETITIVE DNA.

Most of the plant and animal genomes consist largely of repetitive DNA.

There are two main types of repeated sequence:

Tandem repeats: These are common sequences found anywhere in genome

Interspersed repeats: These are repetitive sequences found in between genes.

TRANSPOSABLE ELEMENT

"A transposable element is a bit of DNA that can move from place to place in an organism's genome....”

discovered by Barbara McClintock in 1953.

These elements are also called as Jumping genes / Mobile genetics elements / Controlling elements/

Transposons / Insertion Sequences / Selfish DNA / Roving genes / Cassettes

They are of following types:

1. Autonomous: where transposase gene is functional and carry out mobilization and

2. Non autonomous: where transposase gene is not functional

AC – DS System

In particular, Barbara was analyzing genes that control the color of the aleurone layer of the endosperm. The

genes that she was following were located on the short arm of chromosome 9 of corn and were involved in the

development of the color of the seed.

The grains in the maize kernel are variedly coloured, Patched and white. This variation in colour is due to

shifting of Transposable elements Ac (Activator) and Ds

(Dissociator):

a. pigments produced by gene leading to the

coloured grains

b. Gene function blocked by insertion of Ds element

in between lead to loss of pigment and hence

white in colour

c. Removal of the Ds element with the help of Ac

element lead to formation of Patched colouration

in seeds

Hence the differential colours are due to actions of Ac and

Ds elements in Maize.

UNIT II:

GENE CONCEPT (05 Marks)

Gene is the basic functional unit of life.

The concept of gene was first introduced by Mendel and he termed them as hereditary factors or elements.

Wilhelm Johansen coined the term Gene in 1909 to describe heritable factors responsible for the transmission and expression of a given biological character.

This concept was known as classical gene structure. It states that

1. Genes determine physical as well as physiological characteristics.

2. Genes are present on the chromosome, and on a single chromosome there are many genes.

3. Genes occupy a specific position on a chromosome which is called as locus or loci and genes are arranged in single linear order.

MODERN GENE CONCEPT (05 Marks)

Seymour Benzer (October 15, 1921 – November 30, 2007) proposed the modern concept of gene. He has been defined gene as follows:

1. Cistron The portion of DNA specifying a single polypeptide chain is termed as cistron, which is a synonym for the termed, the gene of physiological function.

2. Muton There are many positions or sites within a cistron where mutations can occur.

3. Recon Sometimes crossing over or recombination occure in a cistron and this provides still, other sub-divisional concept of the cistron, namely the recon.

CENTRAL DOGMA (05 Marks)

Protein synthesis is the key physiological process in all living organisms. Crick, 1958 proposed that genetic information flows from nucleic acid to the protein through two-step process as follows –

Duplication Transcription Translation

DNA RNA Protein

In 1968, Berry Commoner revised it in circular flow of information.

DNA RNA

RNA Protein

The protein synthesis is carried out in following two major steps –

A) Transcription: Formation of RNA by copying genetic information from DNA to RNA

B) Translation: Formation of a Chain of Amino acid (Peptide/Protein) by translating information from mRNA with the help of tRNA and rRNA in to peptide.

TYPES OF RNA (05/12 Marks)1. Messenger RNA: Messenger RNA (mRNA) is

synthesized from a gene segment of DNA. The messenger RNA carries the code into the cytoplasm where protein synthesis occurs.

2. Ribosomal RNA: In the cytoplasm, ribsomal RNA (rRNA) and protein combine to form a nucleoprotein called a ribosome.

3. Transfer RNA: Transfer RNA (tRNA) contains about 75 nucleotides, three of which are called anticodons, and one amino acid. The tRNA reads the code and carries the amino acid to be incorporated into the developing protein. There are at least 20 different tRNA's.

PROPERTIES OF GENETIC CODE (12 Marks)The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells. Specifically, the code defines a mapping between tri-nucleotide sequences called codons. The genetic code is elucidated by Nirenberg, Khorana and Holley in the year of 1959.

The genetic code has following properties –

1. Triplet Nature: Each codon consist of three bases e.g. UUU for Phenyl alanine.

2. Non Overlapping: The codon are read by triplets without any overlapping. Codons are purely non

overlapping. Change in a base can affect more than one codon

3. Non Punctuating: Genetic code is non punctuating, there is no punctuating codon in between.

4. Degeneracy (Synonyms): One amino acid is coded by many (determined) number of codons. The Codons

are degenerate, they do not posses independent coding by them only, the same amino acid can be coded by

other codon. These codon are called as Synonymous Codons.

5. Non Ambiguous: A particular codon will always code for a specific amino acid. There is no ambiguity

(doubt) in the meaning of the codon.

6. Universal: All the living organisms are having same meaning of the code. The genetic code is valid for

microbes, plants and animals. i.e. AGA codon is coding for arginine in all the organisms.

7. Wobbling : Wobble hypothesis proposed by Dr. F. H. C. Crick (1965) According to this hypothesis, only

the first two bases of the codon have a precise pairing with the bases of the anticodon of mRNA, while the

pairing between the third bases of codon and anticodon may Wobble (non specific).

RNA PROCESSING

The initial primary transcript synthesized by RNA polymerase II during transcription undergoes several processing steps before a functional mRNA is produced. It happens only in eukaryotic cells and called as RNA processing, which includes three major processes:

1. 5′ capping The 5′-Cap is added to nascent mRNA 2. 3′ polyadenylation: A tail of Adenine is added at

3’ end3. RNA splicing: During the final step in formation

of a mature, functional mRNA, the introns (non coding region) are removed and exons are spliced together.

RIBOSOME AS TRANSLATION MACHINEThe structure of the ribosome consists of a binding site for mRNA and three binding sites for tRNA:

1. For tRNA, the P site (peptidyl-tRNA site) carries the growing polypeptide chain, while

2. A site (aminoacyl-tRNA site) holds the tRNA that carries the next amino acid that is to be added to the growing chain.

3. E site (exit site) is the site where discharged tRNAs leave the ribosome.

ENDOMEMBRANE SYSTEM

The endomembrane system is composed of the different membranes that are suspended in the cytoplasm within a eukaryotic cell. In eukaryotes the organelles of the endomembrane system include: Nuclear Envelope, Endoplasmic Reticulum, Golgi Apparatus, Lysosomes, Vacuoles, Vesicles, Endosomes and the Cell Membrane.

Exit Tunnel

UNIT – IIIOPERON CONCEPT (12 Marks):

Operon is the model of regulation of gene expression proposed by F. Jacob and J. Monod in 1961. This model is popularly known as Operon Model. Accordingly, Operon is define as a Cluster of Genes as a single unit to regulate protein synthesis. It consisted of –

A. Regulatory Genes: These are the genes coding for regulatory proteins which may be Repressor or Activator. It controls Transcription of the gene.

B. Regulatory sequences:

i. Operator: The sequence of the DNA regulating the switch on and off mechanism by interacting with regulatory proteins like repressor or inducer

ii. Promoter: It is the part of DNA providing landing platform for transcription enzyme RNA polymerase.

C. Structural Genes: These are the genes synthesizing the actual proteins by transcription and translation of own information.

LAC OPERON

It is the proposed mechanism of gene expression in Bacteria E. coli. In different condition / availability of sugar how bacteria are producing different enzymes was explained by this model.Being simplest sugar glucose is preferred by the bacteria for utilization. But when glucose is not available for metabolism then bacteria will search for other kind of sugars. It has own mechanism to utilize Lactose, which is disaccharides sugar made up of Glucose and Galactose.To utilize this sugar (Lactose) bacteria needs to synthesize different enzymes like β Galactosidase (lac Z), β Galactioside Permease (Lac Y) and β – thiogalactoside acetylase (Lac A).

Naturally when a bacterium is not in need to utilize Lactose these genes are switched off with the help of Repressor Protein synthesized by Regulatory Gene

BRITTON DAVIDSON’S MODEL (05 marks)

It is also known as Operon Operator Model or the Gene-Battery Model. It is a hypothesis for the regulation of protein synthesis in eukaryotes. Proposed by R. J. Britten and E. H. Davidson in 1969, the model postulates four classes of DNA sequences: an integrator gene, a producer gene, a receptor site, and a sensor site.

PROTEIN STRUCTURE (05 /12marks)

The shape of a protein is very important for its function. Following are four levels of protein structure: primary, secondary, tertiary, and quaternary.

Primary: The unique sequence of amino acids in a polypeptide chain is its primary structure.

Secondary: The local folding of the polypeptide in some regions gives rise to the secondary structure of the protein. The most common are the α-helix and β-pleated sheet structures.

Tertiary: The unique three-dimensional structure of a polypeptide is its tertiary structure.

Quaternary: In nature, some proteins are formed from several polypeptides, also known as subunits, and the interaction of these subunits forms the quaternary structure.

PROTEIN FOLDING (5 marks)

Protein folding is the process by which a protein structure assumes its functional shape or conformation. To fold proteins following interactions are important –

1. Hydrogen Bond: 2. Disulfide Bond: 3. Hydrophobic Interactions: 4. Ionic Interaction:

PROTEIN TARGETING (5 marks)

Protein targeting or protein sorting is the biological mechanism by which proteins are transported to the appropriate destinations in the cell or outside of it. Proteins can be targeted to the inner space of an organelle, different intracellular membranes, plasma membrane, or to exterior of the cell via secretion. This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases.

Targeting signals are the pieces of information that enable the cellular transport machinery to correctly position a protein inside or outside the cell. There are two types of targeting peptides –

1. The presequences and

2. The internal targeting peptides.

PROTEIN SORTING (5 marks)

3. Many different proteins are synthesized on the rough endoplasmic reticulum (RER) and transported to the Golgi apparatus in transport vesicles. From the Golgi apparatus these proteins are then sent to their proper locations in the cell.

4. To insure that these different proteins are sent to their proper locations, they must be sorted, one from the other, in the Golgi apparatus. Thus one function of the Golgi apparatus is protein sorting.

PROTEIN TRAFFICKING (5 marks)

The Golgi apparatus is involved in the sorting and trafficking of proteins produced within a cell. Proteins translated within the rough endoplasmic reticulum are transferred to the Golgi. From there they are modified and packaged into vesicles for distribution.

UNIT IV:

RESTRICTION ENZYMES (5 marks)These are “molecular scissors” cutting DNA of living organisms, such enzymes are called as Restriction Enzymes.Nomenclature Restriction Enzymes The names of the enzymes are derived from the sources i.e. Genus- first letter capitalized, Species - second and third letters (small case, Italicized), and Additional letters from “strains” and roman numeral designates different enzymes from the same bacterial strain, in numerical order of discovery. Example: EcoRI – E: Escherichia, Co: coli, R: R strain, I: first enzyme discovered from Escherichia coli RClassification of Restriction Enzymes: Restriction enzymes are of following types –

PROPERTIES OF GOOD VECTORS (5 marks)Vectors are the gene vehicle to transfer gene from one organism to the other. Many organisms including microbes, plants and animals. These are also called as Gene Taxies/Gene Carrier. They are DNA with following properties : Extra-chromosomal, Double Stranded, Circular, Autonomous DNA Possesses origin of replication (Ori) Site & Possess the Marker Genes.

PLASMIDS: pBR 322: This is a Plasmid discovered by Boliver & Rodriguez hence named as p(Plasmid) B(Boliver) R(Rodriguez). It is derived from bacteria E. coli. This plasmid is of 4362 bp DNA. The marker genes found in this plasmid are Tetracyclin & Ampicilin. There are around 12 restriction sites in the marker genes.

Restriction Enzymes Restriction ExonucleaseRestriction EndonucleaseRestriction Endonuclease Type IRestriction Endonuclease Type IIRestriction Endonuclease Type III

BACTERIOPAHAGES: Bacteriophage is generally called as phage. It is a virus that infects and replicates within bacteria. The plasmids are vectors for cloning small pieces of DNA.Bacteriophage lambda vectors posses for properties to serve as a gene vehicle in genetic engineering :

1. They could complete their life cycles even if foreign DNA was inserted into a portion of its genome.

2. The genome size is Genome 48502 BP 3. They contain stuffer DNA (certain regions of the virus genome which

is not essential) to get replaced by Gene Of Interest to transfer. 4. The marker genes like gam and red are present.5. Regulator genes are also present6. Restriction sites present in the DNA allows Gene Of Interest to insert in to it.

COSMID :Cosmids are combination of plasmids and Phages Lambda DNA which includes a. Cos region, b. marker gene and c. restriction sites. The long DNA (45 kbp) can be introduced in other organism by using cosmid. It infects like bacteriophage. Its selection and amplification is easy. Generally it is used in making gene library.

PCRThe polymerase chain reaction (PCR) is a biochemical technology in molecular biology used to amplify a single of a piece of DNA generating thousands to millions of copies of a particular DNA sequence. This technology was developed in 1983 by Kary Mullis.Following are fundamental steps in PCR:1. Denaturation: Heating the reaction to 94–98 °C for 20–30

seconds. It causes DNA melting of the DNA template yielding single-stranded DNA molecules.

2. Annealing: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. The polymerase binds to the primer-template hybrid and begins DNA formation.

3. Extension/elongation: The temperature at this step brought to 72 °C which is optimum for activity of enzyme Taq Polymerase. At this step the Taq DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction. Under the optimum conditions, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

GENOMIC DNA LIBRARY

Genomic DNA library indicates that this is the storage of complete genomic DNA in a library. The preparation of this library involves following steps –

1. Extraction of entire genomic DNA

2. Restriction Digestion

3. Separation of fragments of equal size

4. Insertion of the fragments in vector

5. Vector containing genomic DNA cloned in host

C- DNA LIBRARY

C- DNA library is also known as copy or complementary DNA library. As its name indicates, this library is prepared from the mRNA. The preparation of this library involves following steps–

1. Isolation of mRNA2. Priming of mRNA with oligo dT from the 3’ end of RNA3. Reverse Transcriptase synthesizes DNA on mRNA forming RNA-DNA hybrid4. Treatment with alkali degrades RNA from the hybrid5. Single stranded DNA copied by DNA polymerase making it double stranded in continuation with the hook6. The hook is single stranded DNA, which is cleaved by the enzyme known as S1 – Nuclease. 7. These double stranded DNA are cloned in vectors to deposit in the gene library.

Chemical method of Gene Transfer:Chemical compounds used to facilitate DNA transfer are often named as transfection reagents. There are four major types of chemical reagents that are available: calcium phosphate, DEAE-dextran, cationic lipid, and cationic polymer.

Electroporation method of Gene Transfer:Electropermeabilization is a significant increase in the permeability of the cell plasma membrane caused by an externally applied electrical field.

Gene Gun Method: A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics.

AGROBACTERIUM MEDIATED GENE TRANSFERThis method involves the use of a plant pathogen called Agrobacterium tumefaciens, which causes crown gall disease in many species. This bacterium has a plasmid known as Ti Plasmid, or loop of non-chromosomal DNA, that contains tumor-inducing genes (T-DNA “Transfer”), along with additional genes that help the T-DNA integrate into the host genome.Agrobacterium is considered as a natural DNA

delivery system. It cuts its own T-DNA and introduce it to the cell it is infecting.